A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) is now known to be a major controlling factor of platelet production. The studies necessary to determine the chemical characteristics, site of production, mechanism(s) of action, mechanism(s) that sense the need for TSF release and action have not been performed because of the lack of suitable assay techniques and the availability of a stable purified source of TSF. Research described in this proposal will provide new assay procedures and a suitable supply of TSF for further studies. The objectives of this proposal are to: 1) continue production of TSF and establish its chemical and biological nature, 2) determine the amino acid sequence and clone the gene for TSF, 3) characterize mRNA from human embryonic kidney (HEK) cells, 4) measure the efficacy of TSF in bone marrow suppressed animals, 5) develop a radioimmunoassay for TSF, 6) measure ploidy of megakaryocytes in animals after TSF treatment, 7) establish TSF combining sites on megakaryocytes, 8) clarify effects of hypoxia on murine metakaryocytopoiesis, 9) establish cell-lines from tumors that produce TSF, 10) continue TSF testing in body fluids of patients with platelet production disorders, and 11) begin new studies on genetic transfer of TSF. These objectives will be pursued utilizing a variety of physical chemical and immunological methods: 1) sera from thrombocytopenic animals and culture media from HEK cell cultures will be utilized for TSF production; 2) antisera will be raised in animals by use of conventional and monoclonal antibody technology; 3) purification of TSF from a variety of sources will be made by HPLC and immunological techniques; 4) proteolytic cleavage will be employed to generate fragments of TSF for sequence determination; 5) amino acid sequences of TSF will be used to generate DNA probes to identify TSF producing clones in a human cDNA library currently available at Abbott Laboratories; 6) mRNA will be extracted from HEK cells and separated from other nucleic acids by affinity chromatography on oligo-dT cellulose columns; 7) mRNA will be injected into frog oocytes and TSF production measured; 8) mice will be exposed to hypoxia and/or 89Sr to determine the relative roles of the spleen and bone marrow in megakaryocytipoiesis; 9) mice and dogs will be injected with TSF and their platelet production measured in order to determine the efficacy of TSF; and 10) retroviral-mediated gene transfer in an animal model will be attempted. These studies will provide the necessary information to predict the mode of action of TSF and it mechanisms of action. Accomplishment of the goals listed in this proposal will make available recombinant TSF for treating patients with platelet production disorders, in accelerating platelet recovery in thrombocytopenic patients following radio- or chemotherapy-induced bone marrow depression, bone marrow transplantation, and AIDS.